1. Field of the Invention
The present invention relates to the isolation of a DNA from locus 4p16 of chromosome 4, which encodes a novel member of a family of protein kinases that specifically phosphorylate the activated forms of G protein-coupled receptors, thereby desensitizing the receptor and blocking further signal transduction.
2. Background of the Invention
2.1 The G Protein-Coupled Receptor Kinases.
The G protein-coupled receptors mediate responses by guanine nucleotide-binding regulatory proteins (G-proteins) to a wide range of extracellular stimuli, including hormones, neurotransmitters, peptides, odorants, and light. Evidence has shown that G protein-coupled receptor desensitization may be caused by specific phosphorylation of the receptor. Palczewski and Benovic, TIBS 16:387-391 (1991). Furthermore, several investigators have proposed that phosphorylation of the receptor protein by highly specific receptor kinases may represent an important and unifying mechanism serving to dampen receptor function in the presence of persistent or excessive stimulation. Lorenz et al., Proc. Natl. Acad. Sci. U.S.A. 88:8715-8719 (1991). Biochemical data have demonstrated that the G protein-coupled receptor kinases are unique in their ability to recognize and phosphorylate only the stimulus-modified or activated conformations of the G protein-coupled receptors. Benovic et al., Proc. Natl. Acad. Sci. U.S.A. 83:2797-2801 (1986); Sibley et al., Cell 48:913-922 (1987); Benovic et al., Annu. Rev. Cell Biol. 4:405-428 (1988).
Two specific enzymes from the family of serine/threonine kinases, which mediate stimulus-specific phosphorylation of G protein-coupled receptors have been characterized. Specifically, light activated rhodopsin has been shown to be phosphorylated and inactivated by rhodopsin kinase (ROK), whereas the agonist-occupied .beta..sub.2 -adrenergic receptor (and possibly other G protein-coupled receptors) has been shown to be phosphorylated and desensitized by the .beta.-adrenergic receptor kinase (.beta.ARK). Palczewski and Benovic, TIBS, supra (1991).
Currently, rhodopsin kinase has only been cloned from cattle (Lorenz et al, supra (1991)) and has yet to be mapped in mouse or man. However, two forms of .beta.ARK have been identified to date, .beta.ARK and .beta.ARK2. .beta.ARK has been cloned from cattle (Benovic et al., J. Biol. Chem. 269:9026-9032 (1987)), rat (Arriza et al., GENBANK accession number M87854) and man (Benovic et al., FEBS Lett. 283:122-126 (1991)), while .beta.ARK2 has been cloned only from cattle (Benovic et al., J. Biol. Chem. 266:14939-14946 (1991)) and rat (Arriza et al., GENBANK accession number M878555). In addition, two new members of the family of G protein-coupled receptor kinases (GPRK), GPRK-1 and GPRK-2, have been recently cloned from Drosophila (Cassill et al., Proc. Natl. Acad. Sci. U.S.A. 88:11067-11070 (1991)). Although cloned from retina, the receptor kinases were ubiquitously expressed.
Both rhodopsin kinase and .beta.ARK specifically phosphorylate the activated form of the corresponding receptor, increasing its ability to interact with retinal arrestin or .beta.-arrestin, respectively. However, it is interesting to note that rhodopsin and .beta.-adrenergic receptors can be properly phosphorylated by each other's kinase, albeit at lower affinity, and only in their active state. Cassill et al., supra (1991).
Phosphorylation alone has been shown to result in only a slight decrease in receptor activity, but causes the receptors to be high-affinity substrates for the arrestin proteins. The phosphorylation of the receptor by the kinase has been shown to be a prerequisite for arrestin binding; however, binding of the arrestins to the receptors then blocks further activation of the respective signal transduction pathways. In other words, the enzymatic chain of events effectively "turns off" the active state of the receptor by preventing the receptor from coupling to the G-protein. Gassill et al., supra (1991).
It is apparent from the foregoing that there is a long-felt need in the art to understand the biochemical mechanisms involved in receptor transduction pathways. Clearly, identification and characterization of a novel member of the family of protein kinases that specifically phosphorylate the activated forms of G protein-coupled receptors would significantly advance the art, particularly since the kinases are thought to desensitize the specific receptors, thereby blocking signal transduction. The present invention satisfies this need and provides related advantages in the art.
2.2 Identification of the Huntington's Disease Region.
Huntington's disease is an autosomal dominant progressive neurodegenerative disorder of mid-life onset involving uncontrolled choreic movements, psychiatric disturbance and cognitive decline. The disease is characterized by extensive neuronal cell death, particularly in the caudate nucleus. Martin and Gusella, New Engl. J. Med. 315:1267-1276 (1986).
To date, no biochemical mechanism underlying the expression of Huntington's disease has been explained in either patients afflicted with the disease, or in individuals genetically predisposed to the disorder. Bates et al., Am. J. Hunt. Genet. 49:7-16 (1991). However, it was recognized that molecular genetic methods could be utilized in an indirect approach, to search for a gene linked to Huntington's disease via chromosomal mapping, although no knowledge of the gene product was available. Previously, the more traditional route of searching for an aberrant protein was unlikely to be successful in Huntington's disease, given the complexity of the affected tissue, the strong likelihood of confounding secondary changes, and the lack of availability of brain tissue from patients afflicted with Huntington's disease until late in the course of the disorder. Gusella, Adv. Hum. Genet. 20:125-151 (1991).
As a result of the high penetrance of the Huntington's disease defect and of the relatively long course of the disease after the onset of symptoms, it has been possible to identify very large extended kindreds with many affected members. The strategy for identifying the region of the Huntington's disease defect within the human chromosome has involved using naturally occurring variations in DNA sequence in the human population as high-quality genetic markers for tracking the transmission of particular chromosomal regions through disease pedigrees. Gusella, Adv. Hum. Genet., supra.
The Huntington's disease region was first localized in 1983 by genetic linkage analysis using the DNA marker D4S10 and two Huntington's disease pedigrees, an extremely large Venezuelan family and a smaller 14 member American kindred. The disease gent was mapped to chromosome 4, marking the first instance in which a genetic defect had been mapped to a chromosome using only DNA marker linkage analysis. Gusella et al., Nature 306:234-238 (1983); Gusella et al., Science 225:1320-1326 (1984); see also U.S. Pat. No. 4,666,828, issued May 19, 1987.
As disclosed by Allitto et al. in Genomics 9:104-112 (1991), mapping of the DNA marker D4S10 by dosage studies in Wolf-Hirschhorn syndrome (Gusella et al., Nature 318:75-78, (1985)), by in situ hybridization (Zabel et al., Cytogenet. Cell Genet. 42:187-190 (1986); Magenis et al., Amer. J. Hunt. Genet 39:383-391 (1986); Wang et al., Amer. J. Hum. Genet. 39:392-396 (1986); Landegent et al., Hunt. Genet. 73:354-357 (1986)), and by somatic cell hybrid analysis (MacDonald et al., Genomics 1:29-34 (1987); Smith et al., Amer. J. Hum. Genet. 42:335-344 (1988)) has placed the marker in chromosomal locus 4p16. In addition, somatic cell hybrid panels were constructed to permit the mapping of DNA probes to either the proximal or the distal portions of 4p16.3. MacDonald et al., supra, (1987); Smith et al. supra, (1988).
The mapping of the Huntington's disease marker, D4S10, near the telomere of 4p has created the impetus for constructing detailed genetic and physical maps of the terminal segment of 4p. Allitto et al., supra. (1991). However, the critical issue in isolating the Huntington's disease gene has been to define which portion of the physical map contained the defect. In several other disorders, the identification of a disease gene has been facilitated by the observation of a physical rearrangement in the candidate region. Unfortunately, no such physical alteration has been reported for the region of the Huntington's disease defect. Gusella, Adv. Hum. Genet., supra.
Consequently, the only means available to establish the position of the defect on the physical map was to analyze Huntington's disease affected families in which recombination has occurred between the disease gene and markers of known position in 4p16.3. Gusella, Adv. Hum. Genet., supra. Attempts to isolate the Huntington's disease gene based on its position in the proximal portion of the terminal cytogenetic subband, 4p16.3, of chromosome 4, were frustrated by apparently contradictory recombination events.
However, more recent multipoint linkage mapping using a proximal marker has established that the Huntington's disease genetic region is located distal to D4S10 within 4p16.3, which constitutes 3% of the cytogenetic length of chromosome 4 or approximately 0.2 % of the total genome (Gilliam et al., Cell 50:565-571 (1987)). Moreover, using multi-allele polymorphisms to assess the consistency of the haplotypes present in the Huntington's disease region of the chromosome, MacDonald et al. have found that about one third of all Huntington's disease chromosomes derive from one primordial haplotype, which is most consistent in the proximal portion of the internal candidate region. See, Nature Genet. 1:99-103 (1992). Thus, haplotype analysis has indicated that a 500 kb segment between D4S180 and D4S182 was the most likely site of the genetic defect (MacDonald et al., supra (1992)).
It is apparent from the foregoing that there has remained a long-felt need in the art for the determination of the biochemical mechanism which results in Huntington's disease in genetically predisposed patients. Clearly, there has been great interest in the identification of a gene related to Huntington's disease, as well as a determination of its sequence and expression product to provide reliable probes for the detection of the disease in a patient. Such advances would permit a direct experimental approach to identifying the fundamental mechanisms involved in the activation of this devastating disease. Therefore, identification and characterization of a gene related to Huntington's disease would significantly advance the art.